1a.Objectives (from AD-416)
1. To conduct a genome-wide expression profiling of small RNAs to identify sets of small RNAs specifically expressed during germination of cysts, growth in broth-shake culture and during infection of roots.
2. Locate and characterize the genome-wide distribution of small RNA-generating loci
3. Characterize the effect of DCR, RDR and AGO mutations on small RNA populations.
4. Development of a Phytophthora small RNA database and a public website and integration with other Phytophthora genome resources

1b.Approach (from AD-416)
Our project will start with small RNA analysis in P. sojae lifestages and infected roots, followed by functional analysis of P. sojae mutants. First, we will document the small RNA repertoire in pure P. sojae lifestages and in infected soybean roots by deep sequencing on the Illumina platform. Next we will complement small RNA analysis using tilling mutants in the key enzymes DCR and RDR which are responsible for synthesis of small RNAs. Select mutants and selected mutants will be analyzed in the mycelial lifestage and in life stages in which a phenotype is expressed. Throughout, the existing preliminary Phytophthora small RNA Database will be expanded, updated and improved. This database will provide a repository for sequences of small RNAs cloned from various Phytophthora spp., genotypes and tissues. The database will integrate tools to assist in miRNA and siRNA identification and analysis.

3.Progress Report

The overall goal of our research is to identify the genetic mechanisms that enable oomycete pathogens to overcome host defenses, using the soybean pathogen Phytophthora sojae as a model. This proposal focuses on genome-wide analysis of small RNAs expressed during different life stages and during infection of soybean by the pathogen Phytophthora sojae. Three small RNA libraries from the Phytophthora species P. infestans, P. sojae and P. ramorum were produced and sequenced using high throughput sequencing technologies. Two small RNA size classes, 21 and 25 nucleotides in size, were identified in each library. A database and genome viewer were developed to facilitate analysis of the small RNA component in Phytophthora. Putative RNA silencing biogenesis genes were tentatively identified in the P. sojae, P. infestans, and P. ramorum genomes: Two distinct dicer genes, named dicer-like 1 and dicer-like 2, were cloned from Phytophthora, and their gene structure and evolutionary relationships to other known dicers were described. The gene structure for another small RNA biogenesis effector, RNA-dependent RNA polymerase, was also determined. A special cllass of small RNAs termed micro RNAs was discovered. Eight candidate micro RNA genes from one gene family were identified in P. sojae, P. ramorum, and P. infestans. We are now evaluating using artificially designed micro RNAs as tools for targeted, stable silencing in Phytophthora. To date, micro RNA pathways have only been described for animals and plants and are newly characterized in the Stramenopile branch of the tree of life. Thus, this is the first report of presence of micro RNAs in a new branch of the tree of life. Artificial micro RNAs show promise as a novel tool for research and potentially management of Phytophthora.